RESULTS AND DISCUSSION 1. Fermentation stage

Aspergillus niger grew well on medium containing SB waste material as a substrate and a rapid mycelial growth was recorded at the beginning of the cultivation period followed by a slow growth phase (Table I). The initial (first 3 days) average growth rate was 0.19 g/flask/day and 0.02 g/flask/day during the second week. It increased again thereafter and the total biomass produced on medium supplemented with RP was 20% higher than that of 1.0 g/flask obtained [7] on the medium without RP. Mycelial growth and titratable acidity production paralleled in the first half of the process resulting in a maximum solubilization (76%) of the rock phosphate. However, after this period the fungus started to produce spores, indicating adverse conditions for acid productivity. This caused a slow decrease in titratable acidity with a corresponding decrease in soluble P production.

Biomass growth on the lignocellulosic substrate tested in this study was higher than that under liquid culture conditions using the same strain of A. niger [13]. Other authors applying sugarcane bagasse [23] and vinasse [24] have reported similar results. The most likely explanation is the the presence of

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TABLE I. MYCELIAL GROWTH, TITRATABLE ACIDITY AND ROCK PHOSPHATE SOLUBILIZATION BY Aspergillus niger CULTIVATED ON SUGAR BEET WASTE _____________________________________________________________________

Titratable Phosphate

Time Biomass acidity Citric acid (% conc. Soluble P/

(days) (g/flask) (mmol/l) of total acidity) ( g/ml) total P (%) _____________________________________________________________________

3 0.57+0.12 38.2+1.4 100 47.4+1.6 12 6 0.68+0.08 60.6+0.7 98 172.6+0.8 44 10 0.77+0.05 72.2+0.6 92 292.2+2.3 76 15 0.89+0.05 58.8+1.1 87 276.8+3.1 71 20 1.20+0.10 48.1+0.2 71 224.0+0.9 58 _____________________________________________________________________

TABLE II. COMPOSITION OF SUGAR BEET WASTE¹ SUSPENSION TREATED WITH Aspergillus niger AFTER A 20-DAY CULTIVATION

__________________________________________________________________

Cellulose Hemicellulose Lignin Mineralization (%) (%) (%) pH (%) __________________________________________________________________

Initial 29.0 23.0 5.0 7.2 - 20 d 17.0 4.2 4.2 3.2 69

__________________________________________________________________

1 Total carbon: 55%; Oxidizable carbon: 16%; Total nitrogen: 1.7%

sufficient amounts of nutrients in the initial period of cultivation and some lignocellulolytic activity (Table II) bearing in mind the ability of A. niger to degrade such kind of substrates [23]. The specific conditions provided by the microorganism/air/water interface during this type of cultivation [23], determined a level of acidity, which was sufficient to overcome the neutralizing effect of RP and its solubilization.

It should be noted that the measured phosphate concentration in the solution probably

corresponded to the amount that was not consumed by the mycelium. The addition of RP to the cultivation medium affected the behavior of A. níger, particularly its growth and citric acid production.

Although a high percentage of mineralization of SB waste was achieved, a careful assessment and further studies should be done in order to find a better compromise between the level of degradation and concentration of soluble phosphate before the resulting system is applied in soils with different characteristics.

3.2. Soil-plant experiment

Dry matter responses and P content of shoots of non-mycorrhizal and mycorrhizal T. repens after the addition of microbially-untreated SB with or without RP and previously cultivated SB (with or without RP) by A. niger are presented on Fig. 1 and Fig. 2, respectively.

51 Fig. 1. Shoot dry weight of Trifolium plants grown for 42 days as affected by fermentation-resulting products and mycorrhizal inoculation. * Denotes significant differences in mycorrhizal response.

I Denotes significant differences in RP response.

Fig. 2. Shoot P content of Trifolium plants grown for 42 days as affected by fermentation-resulting products and mycorrhizal inoculation. * Denotes significant differences in mycorrhizal response.

I Denotes significant differences in RP response.

The addition of SB alone or in combination with RP in the absence of A. níger did not have a significant effect on plants weight of either, non- and mycorrhizal soil-plant system. However, plant P acquisition was favored by G. deserticola in the respective treatments. The addition of microbially precultivated SB without RP increased plant weight of T. repens and almost doubled its P content compared with the control and SB with RP treatments. These effects could be related to the low pH of the mixture due to the presence of citric acid. Although the soils in Southern Spain have a high phosphate-fixing capacity, it was reported recently that the addition of citric acid increased phosphate concentrations in solutions of alkaline soils, this effect being detectable even after 140 days [25].

A significantly high growth and P shoot content was found in plants grown in soil amended with the lignocellulosic substrate with RP and A. niger mixture. This observation was even more pronounced in plants inoculated with G. deserticola. The triple combination between A. niger, R. trifoli and G.

deserticola caused a response in plant growth and P uptake of both non-mycorrhizal and mycorrhizal plants, respectively, but the inclusion of RP in the system enhanced this effect. The assumption that A. niger can provide additional nutrient amounts derived from the mycelium cannot explain the lower plant growth and P uptake in the treatment without RP and AM inoculation. This work confirms the important role of mycorrhizal fungi and particularly their synergistic effect in combination with P-solubilizing microorganisms, as reported previously [26].

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The effects of certain combinations of treatments produced a lowering in the SA of the clover plants (Fig. 3). This lowering was clearer in AM-inoculated plants, particularly when RP was a component of the fermentation mixture. It could be argued that the treatments having the lower SA values suggest that the plant is using extra ³¹P released from otherwise unavailable P sources [14]. The phosphate-solubilizing activity of A. niger, could in part release P ions from the added RP and the AM external mycelium was transferring to the plant ³¹P released from RP particles, therefore inducing a lowering in the ³²P/³¹P ratio [26, 27]. Therefore, the lowering in the specific activity is indicating solubilization of RP by the microbial activities applied in the reported biotechnological approach.

Fig. 3. Specific activity of Trifolium plants grown for 42 days as affected by fermentation resulting products and mycorrhizal inoculation. * Denotes significant differences in mycorrhizal response.

I Denotes significant differences in RP response.

4. CONCLUSIONS

The biological solubilization of RP using the metabolic, acid-producing activity of the filamentous fungus A. niger during its cultivation on sugar beet waste material and further application of the resulting mixture appears to be effective in a neutral, calcareous soil as shown by the isotopic (³²P) dilution technique. The interaction between a biotechnological practice (triple inoculation with R. trifoli, A. niger and G. deserticola) and a low-input technology (RP addition to the system) demonstrates the potential application of such combined approaches in improving sustainable nutrient supply to plants.

ACKNOWLEDGEMENTS

These studies were carried out under the auspices of the research agreement No. SPA-7418 with the International Atomic Energy Agency.

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